hHCN4基因修饰的大鼠BMSCs与心肌细胞共培养分化为心肌样细胞的实验研究

Differentiation of bone marrow mesenchymal stem cells transfected HCN4 gene into cardiomyocyte-like cells after being co-cultured with cardiomyocytes

目的将超级化环化核苷酸门控阳离子通道4(hHCN4)基因修饰的大鼠骨髓间充质干细胞(BMSCs)与乳鼠心肌细胞(CM)进行共培养,观察其细胞连接功能及起搏功能。方法分离培养乳鼠CM并进行细胞标记,将已标记的CM分别与大鼠BMSCs、hHCN4+BMSCs及hHCN4-BMSCs混合培养。共培养4d后检测各组细胞的自发搏动频率,荧光显微镜下观察两类细胞的空间位置关系,Western blotting检测缝隙连接蛋白43(Cx43)的表达,透射电镜观察细胞间缝隙连接的形成情况。结果共培养4d后,心肌的自发搏动频率明显增高。荧光显微镜下观察发现存在空间位置上相邻的BMSCs和CM。Western blotting结果显示,hHCN4+BMSC与CM共培养组Cx43表达明显高于hHCN4-BMSCs与CM共培养组。透射电镜观察显示,hHCN4+BMSCs可与相邻细胞形成指状交错的连接。结论 hHCN4基因修饰的大鼠BMSCs可与CM建立有效的电机械连接,其自发起搏功能更接近CM,为进一步进行生物起搏治疗提供了实验依据。

Objective To explore the gap junction function and pacing function transformed myocardial cells, rat bone marrow mesenchymal stem cells （BMSCs） transfected by hyperpolarization activated cyclic nucleotide-gated cation channel 4 （HCN4） gene were co-cultured with neonatal rat cardiomyocytes （CM）. Methods After being labelled with DAPI, neonatal rat CM were co-cultured with BMSCs, HCN4＋ BMSCs or HCN4- BMSCs for 4 days, respectively. The spontaneous beating frequency of cells in each group was then detected, and the anatomical location of two types of cells was observed by fluorescence microscopy. Western blotting was used to detect the expression of connexin 43 （Cx43）, and the intercellular gap junction was observed by transmission electron microscopy. Results The spontaneous beating rate of ventricular myocardium was significantly higher after being cultured. The anatomical location of BMSCs was found to be adjacent to CM under fluorescence microscope. Western blotting showed that Cx43 expression was significantly higher in HCN4^＋ MSC co-cultured with CM group than in HCN4^- BMSCs cultured with CM group. Transmission electron microscopy revealed that HCN4^＋ BMSCs formed to be interlaced with adjacent CM. Conclusion The rat BMSCs transfected by HCN4 gene may establish an effective electro-mechanical connection with cardiomyocytes, the spontaneous pacing function is close to that of CM, and the result provides an experimental basis for promoting biological pacemaker therapy.